The present invention relates generally to a method for measuring binding assays and more particularly to measuring binding assays carried out with different size particles wherein the binding assay sample is run through a flow cytometer without separating the sample from the marking agent.
Flow cytometry methods have been developed over time to a point where a single file stream of cells or particles passes through a laser beam. This laser beam can excite any flourescent chemicals present in or on the particles. The emission of the fluorescent chemicals when struck by the laser and also such other properties as light scatter due to the particles passing through the laser beam are measured by detectors. This technique combined with deflection plates as described in a 1973 article (A New Multiparameter Separator for Microscopic Particles and Biological Cells, Steinkamp, et al., Review of Scientific Instrumentation, Vol. 44, No. 9, Sept. 1973, p. 1301) explained how the fluorescence of the subject cells or particles could be used as a criterion to separate them. The article also described the use of uniform plastic microspheres to evaluate the combined methods.
Immunoassay techniques are based upon well known reactions between an antibody and the specific antigen or hapten for that antibody. Often one component is tagged, sometimes with a radioactive isotope or sometimes with a fluorescent tracer material, to quantify the percentage of reagent coming from a sample of unknown composition as compared with a known amount of tagged material. U.S. Pat. No. 4,100,416 showed the use of fluorescent tracer material in a competitive binding situation. Competitive binding assays were explained in U.S. Pat. No. 3,939,350. Analysis of the results of the competitive binding assays in U.S. Pat. No. 4,100,416 was by use of a totally internally reflecting cell. In addition to antibodies and antigens, other reacting systems have been used, such as enzyme and substrate for the enzyme, hapten and antibody for the hapten, or a ligand and its receptor. Finally, the use of immunomicrospheres was suggested (Immunomicrospheres: Reagents for Cell Labeling and Separation, A. Rembaum and W. J. Dreyer, Science, Vol. 208, Apr. 25, 1980, p. 364) wherein the immunomicrospheres would be made fluorescent by the addition of the chemical and then bound to larger magnetic beads. Furthermore the article suggested that antibody-coated fluorescent microspheres would attach themselves to antigen previously coated on the wall of the small capillary tube. The bound microspheres would then be eluted and monitored in a fluorimeter.
Problems still remained however with the above methods. Most methods, except for the totally internally reflecting cells, required a step for separating out the unbound, unreacted fluorescent tagged material from the sample before analysis. This separation step proved to be troublesome because the removal of the unreacted tagged material would shift the equilibrium established at the end of the competitive binding process. Aside from the trouble in making sure all the unreacted tagged material had been separated out, the equilibrium shift of the bound tagged material introduced error into the final results. Errors were introduced into the totally internally reflecting cell measurements by the fact that only the tagged cells bound to the wall of the cell would fluoresce when struck by the laser light deliberately confined to the narrow space near the surface of the cell. Additionally, problems were introduced into the measurement technique by the requirement for confining the laser light near the surface of the cell. Finally, a need existed for a method wherein it would be unnecessary to separate the unbound, fluorescent-tagged material from the sample before analysis in a flow cytometer.